Polyethylene glycol-stabilized lipodisks have emerged as a novel type of lipid-based nanoparticles with high potential as both drug carriers and biomimetic membranes. In this thesis we assess both of these applications, and show how the properties of the lipodisks can be further developed and optimized. Initially, we show that the antimicrobial peptides melittin, alamethicin and magainin 2, in spite of their very different physico-chemical properties and suggested modes of action on membranes, all have high affinity to lipodisks. Using melittin as a model peptide, we confirm a maintained antimicrobial effect of disk-formulated peptides. We also show that melittin dissociates slowly from the disks, resulting in extended drug release and prolonged antibacterial effect. Additionally, we present evidence that the peptide is protected against enzymatic degradation when formulated in the disks. Further, we develop a stable HPLC-MS system with immobilized lipodisks as model membranes. The stability of the system is confirmed by drug partitioning analysis using 15 different drug compounds. We also show how the lipodisk column can be supplemented with cyclooxygenase by in situ incorporation of the protein in the lipodisks. The specific binding of the protein to the disks is confirmed using QCM-D. Finally, by changing the polymer length and applying a new preparation protocol, we have optimized the lipodisks for use as drug carriers and biomimetic membranes. Previous lipodisk studies have been conducted on systems containing PEG-lipids with polymer molecular weights of 2000 or 5000 Da. Also, conventional protocols for the preparation of lipodisks typically require a PEG-lipid concentration of 15 mol% or more. Here we show that stable lipodisks can also be produced using PEG-lipids with a 1000 Da molecular weight polymer and that the use of shorter PEG-lipids dramatically improve the amount of lipodisks that can be immobilized on silica surfaces. Moreover, through the development of a method in which lipid mixtures are sonicated at low temperatures, we produce lipodisks containing as little as 2 mol% PEG-lipid. We present data verifying that these disks are superior to disks with higher PEG-lipid content in terms of their ability to incorporate externally added PEG-lipids functionalized with targeting agents.

A fosfoetanolamina sintética (FO-S), um fosfomonoéster, apresenta relevante atividade antitumoral. Contudo, a utilização de um carreador para encapsular a FO-S em lipossomas poderia favorecer a sua disponibilidade no microambiente tumoral, possibilitando o aumento da sua eficácia. Desta forma, o presente estudo avaliou a eficiência de encapsulamento da FO-S em lipossomas de DODAC e o seu potencial antitumoral. Os lipossomas foram preparados por ultrasonicação e caracterizados físicoquimicamente. A citotoxidade foi avaliada nas linhagens tumorais B16F10 (melanoma murino), Hepa1c1c7 (hepatocarcinoma murino) e Skmel-28 (melanoma humano) e nas células normais HUVEC, após o tratamento com diferentes concentrações dos lipossomas DODAC/FO-S, no tempo de 24 horas. A internalização dos lipossomas e o potencial elétrico mitocondrial foram analisados por microscopia confocal a laser. Adicionalmente, a expressão das proteínas caspases 3 e 8 ativas, receptor DR4, citocromo c, p53, p21, Bax, p27, CD44, CD90, Bcl-2 e ciclina D1 foi quantificada por citometria de fluxo. Para os estudos in vivo, os camundongos C57BL/6J portadores de hepatocarcinoma foram tratados com FO-S, DODAC/FO-S e DODAC, pelas vias intraperitoneal (IP) e intrahepática (IH), durante 20 dias. Os resultados demonstraram que os lipossomas apresentaram aspecto esférico e alta eficiência de encapsulação da FO-S, como também promoveram maior citotoxicidade nas linhagens tumorais estudadas, em comparação com FO-S. Além disto, nas células B16F10 e Hepa1c1c7, ocasionou parada nas fases S e G2/M do ciclo celular. A linhagem Hepa1c1c7 foi a mais sensível ao tratamento com os lipossomas DODAC/FO-S, os quais foram internalizados em até 6 horas e promoveram a diminuição de CD90, CD44, ciclina D1 e Bcl-2, o aumento de p53, p21, p27, Bax e caspases 8 e 3 ativas e a liberação do citocromo c. O aumento significativo das caspases 8 e 3 ativas, expressão do receptor DR4 e a liberação do citocromo c também ocorreu nas linhagens B16F10 e Skmel-28. Os resultados in vivo mostraram que os lipossomas DODAC/FO-S e a FO-S não induziram hepatotoxicidade, nefrotoxicidade e caquexia. Os lipossomas DODAC/FO-S não ocasionaram mielossupressão e hemólise, apresentando menor toxicidade em relação a FO-S, administrada pelas vias IP e IH. Além disto, os tratamentos com DODAC/FO-S (IH) e FO-S (IH e IP) foram efetivos em diminuir o número de células na fase S. Contudo, apenas os lipossomas DODAC/FO-S (IH) reduziram significamente os focos tumorais, aumentando as áreas de necrose, promovendo também o aumento da expressão gênica da p53, ciclina B1 e caspases 8 e 3. O conjunto dos resultados in vivo e in vitro demonstraram que a formulação lipossomal DODAC/FO-S foi capaz de maximizar os efeitos antitumorais da FO-S, ativando as vias intrínsecas e extrínsecas da apoptose / Synthetic phosphoethanolamine (PHO-S) - a phosphomonoester - has shown relevant anticancer effects. However, the utilization of a carrier to encapsulate the PHOS in liposomes can maximize its availability in the tumor microenvironment, allowing an increase in its effectiveness. Thus, the present study has evaluated efficiency of PHO-S encapsulation in DODAC liposomes and its antitumor potential. The liposomes were prepared by ultrasonication and physico-chemically characterized. The cytotoxic effects were evaluated on B16F10 cells (murine melanoma), Hepa1c1c7 cells (murine hepatocellular carcinoma), Skmel-28 (human melanoma) and in endothelial cells HUVEC, after treatment with DODAC/PHO-S liposomes at different concentrations for 24 hours. The internalization of the liposomes and mitochondrial electrical potential were analyzed by confocal laser microscopy. Additionally, the expression of active caspases 3 and 8, receptor DR4, cytochrome c, p53 p53, p21, Bax, p27, CD44, CD90, Bcl-2 and cyclin D1 proteins was quantified by flow cytometry. For in vivo studies, C57BL/6J mice with hepatocellular carcinoma were treated with PHO-S, DODAC/PHO-S and DODAC, by intraperitoneal (IP) and intratumoral (IT) routes for 20 days. The results demonstrated that liposomes presented spherical aspect and high PHO-S encapsulation efficiency, as also promoted high cytotoxic effect - compared with PHO-S. Furthermore, in B16F10 and Hepa1c1c7 cells, the liposomes induced S and G2/M cell cycle arrest. Hepa1c1c7 cells showed greater sensitivity to the DODAC/PHO-S formulation, which were internalized until 6 hours and promoted a decrease in the expression of CD90, CD44, cyclin D1 and Bcl-2, an increase of de p53, p21, p27, Bax and active caspases 8 and 3 and the liberation of cytochrome c. The significant increase in the expression of active caspases 3 and 8, DR4 receptor and liberation of cytochrome c also occurred in B16F10 and Skmel-28 cells. In vivo results showed that DODAC/PHO-S liposomes and PHO-S did not induce nephrotoxicity, hepatotoxicity and cachexia. DODAC/PHO-S liposomes did not cause myelosuppression and hemolysis, presenting lower toxicity in relation to PHO-S - when administered by IP and IT routes. Moreover, treatment with DODAC/PHO-S (IT) and PHO-S (IT and IP) effectively decreased the number of cells in S phase. However, only DODAC/PHO-S liposomes significantly reduced the number of tumor foci, increasing area of necrosis, and also promoting an increase in gene expression of p53, cyclin B1 and caspases 8 and 3. The set of in vitro and in vivo results demonstrated that DODAC/PHO-S liposomal formulation was capable of maximizing the PHO-S antitumor effects, activating the intrinsic and extrinsic pathways of the apoptosis

Visando o tratamento tópico de tumores cutâneos, desenvolvemos micro e nanoemulsões para a co-localização cutânea de paclitaxel e ceramida C6. A nanoemulsão mostrou-se mais eficaz em promover penetração de ambos os compostos nas camadas viáveis da pele, com aumentos de 11,5 (paclitaxel) e 3,5 (ceramida) vezes comparado a uma solução controle, e não reduziu significativamente a viabilidade de equivalentes epidérmicos, sugerindo sua segurança para aplicação tópica. A incorporação na nanoemulsão diminuiu as concentrações de paclitaxel e ceramida necessárias para reduzir a viabilidade de células de melanoma a 50% em 6 a 15 vezes, respectivamente, sendo que a co-encapsulação desses compostos promoveu uma nova redução (4 vezes) nessas concentrações, demonstrando aumento da eficácia decorrente da associação dos compostos. A administração tópica do nanocarreador contendo paclitaxel e ceramida co-encapsulados promoveu desorganização e morte celular em equivalentes cutâneos com melanoma, demonstrando o potencial desta formulação para o tratamento tópico de tumores cutâneos. / Nano and microemulsions were developed in this study for cutaneous co-localization of the active agents paclitaxel and C6 ceramide for topical treatment of skin cancer. The nanoemulsion was more effective in promoting the penetration of the compounds into viable skin layers, with increases of 11.5- (paclitaxel) and 3.5- fold (ceramide) being observed. The nanoemulsion did not reduce the viability of human epidermis equivalents, suggesting its safety for topical application. Incorporation in the nanoemulsion reduced the concentration of paclitaxel and ceramide required to decrease viability of human melanoma cells to 50% by 6 to 15-fold compared with their solutions; co-encapsulation of the compounds promoted a further reduction of 4-fold. Topical administration of the nanocarrier with co-encapsulated paclitaxel and ceramide to skin equivalents with melanoma promoted disorganization and intense cytotoxicity. These results demonstrate the potential of nanoemulsions for association of paclitaxel and ceramide C6 to improve their cytotoxic effect and skin localization.

Στο τομέα της νανοϊατρικής ένας από τους σημαντικότερους στόχους είναι η ανάπτυξη φαρμακευτικών νανοφορέων που θα μεταφέρουν και θα αποδεσμεύουν εκλεκτικά το φάρμακο στον πάσχοντα ιστό. Η χορήγηση δοξορουβικίνης (Dox), για παράδειγμα, εμφανίζει σημαντικά προβλήματα έλλειψης εκλεκτικότητας και συστημικής τοξικότητας. Μία πιθανή προσέγγιση για την περισσότερο εκλεκτική χορήγηση της Dox στους καρκινικούς όγκους είναι η χορήγηση της μετά τον εγκλεισμό της σε μαγνητικά στοχευόμενους νανοφορείς. Σκοπός της παρούσας μεταπτυχιακής εργασίας ειδίκευσης ήταν η μελέτη μαγνητικών νανοφορέων με βάση συμπολυμερή πολύ(μεθακρυλικού οξέος)-g-πολύ(μεθακρυλικής αιθυλενογλυκόλης) (p(MAA-g-EGMA) με διαφορετικά χαρακτηριστικά πολυμερικού κελύφους και ο προσδιορισμός εκείνων των χαρακτηριστικών που προσδίδουν στους νανοφορείς βέλτιστη συμπεριφορά. Πιο συγκεκριμένα, μελετήθηκε η σταθερότητα των μαγνητικών νανοφορέων με διαφορετικό μήκος αλυσίδων πολύ(αιθυλενογλυκόλης) και διαφορετική πυκνότητα αρνητικού φορτίου σε διάφορα μέσα όπως υδατικά διαλύματα χλωριούχου νατρίου (ΝαCl), ρυθμιστικού διαλύματος φωσφορικών (PBS), δοξορουβικίνης καθώς επίσης και σε υδατικά διαλύματα διαφόρων τιμών pH. Μελετήθηκε επίσης η φόρτωση του φαρμάκου σε αυτούς καθώς επίσης και η αποδέσμευση του από τους συγκεκριμένους νανοφορείς σε διάφορα μέσα (νερό, υδατικό διάλυμα PBS και διάλυμα αλβουμίνης σε PBS). Οι νανοφορείς παρασκευάστηκαν μέσω πρόσδεσης του συμπολυμερούς πολυ(μεθακρυλικού οξέος)-g-πολυ(μεθακρυλικής αιθυλενογλυκόλης) (p(MAA-g-EGMA) στην επιφάνεια νανοκρυσταλλιτών Fe2O3 κατά τη διάρκεια ανάπτυξής τους. Η μελέτη της σταθερότητας έγινε με τη μέθοδο της δυναμικής σκέδασης φωτός (DLS). Η μελέτη της φόρτωσης και της αποδέσμευσης του φαρμάκου στους και από τους νανοφορείς έγινε με τη μέθοδο της φασματοφωτομετρίας φθορισμού. Στο πρώτο κεφάλαιο παρουσιάζονται συνοπτικά τα διάφορα είδη νανοφορέων, οι ιδιότητες καθώς και οι εφαρμογές αυτών. Γίνεται επίσης μια σύντομη βιβλιογραφική ανασκόπηση σε ότι αφορά τη φόρτωση και αποδέσμευση φαρμάκων από νανοφορείς. Το δεύτερο κεφάλαιο είναι αφιερωμένο στις τεχνικές και τις μεθόδους που χρησιμοποιήθηκαν στα πλάισια της συγκεκριμένης εργασίας καθώς επίσης και των πειραματικών διαδικασιών.Τέλος, το τρίτο κεφάλαιο αφορά στην παράθεση και τον σχολιασμό των αποτελεσμάτων,τα οποία μπορούν να συνοψιστούν στα εξής συμπεράσματα:  Οι μαγνητικοί νανοφορείς με βάση συμπολυμερή πολύ(μεθακρυλικού οξέος)-g-πολύ(μεθακρυλικής αιθυλενογλυκόλης) (p(MAA-g-EGMA) έχουν ικανοποιητικά 7 χαρακτηριστικά μεγέθους και ζ δυναμικού για παρατεταμένη παραμονή στην κυκλοφορία μετά από ενδοφλέβια χορήγηση, γεγονός που αποτελεί προϋπόθεση για την εφαρμογή τους ως συστήματα εκλεκτικής μεταφοράς (στόχευσης) αντικαρκινικών φαρμάκων.  Οι υψηλές τιμές φόρτωσης της δοξορουβικίνης στους νανοφορείς με υψηλή πυκνότητα ανιονικών φορτίων, λόγω ισχυρότερων ηλεκτροστατικών αλληλεπιδράσεων με το θετικά φορτισμένο φάρμακο αποτελεί σημαντικό πλεονέκτημα των νανοφορέων αυτών σαν συστήματα χορήγησης δοξορουβικίνης.  Η αύξηση του ρυθμού αποδέσμευσης της δοξορουβικίνης από τους νανοφορείς σε διαλύματα αλβουμίνης με ελάττωση του pH είναι σημαντική καθώς παρέχει τη δυνατότητα μιας σχετικά εκλεκτικής διάθεσης του φαρμάκου στους καρκινικούς όγκους όπου επικρατούν συνθήκες χαμηλότερου από το το φυσιολογικό pH. Συμπερασματικά, τα αποτελέσματα που λήφθηκαν δικαιολογούν την περαιτέρω μελέτη των μαγνητικών νανοφορέων δοξορουμπικίνης για την καταλληλότητά τους ως φορείς στοχευμένης χορήγησης του φαρμάκου σε καρκινικούς όγκους. / In the field of nanomedicine, one of the most important targets is the development of functional nanoassemblies which will deliver and release selectively the drug to the suffering tissue. For example, the administration of doxorubicin (Dox) displays lack of selectivity and systemic toxicity issues. A possible approach towards a more selective delivery of Dox to the target tissue is its encapsulation at magnetically targeted nanoparticles. The present postgraduate thesis’ aim was the study of magnetic nanocarriers based on copolymers of poly(methacrylic acid)- graft -poly(ethyleneglycol methacrylate) (p(MAA -g- EGMA)) with different structural characteristics and the determination of those characteristics, that impart to the nanocarriers the optimal performance. Specifically, the stability of magnetic nanoparticles, with different chain length of poly(ethyleneglycol) and different density of negative charges, was studied at various media such as NaCl, pH and Dox concentration. The drug loading in the nanocarriers was also studied, as well as its release by the specific nanocarriers at various media (distilled water, PBS and albumin solution in PBS). The nanoparticles were prepared via a self-assembly process of the polymers [poly(methacrylic acid)-graft-poly(ethyleneglycol methacrylate) (p(MAA-g-EGMA)] on the surface of the growing iron oxide nanocrystallites. The stability studies were performed with the use of DLS technique. The study of the drug loading and release from the nanoparticles was followed using the fluorescence spectroscopy. In the first chapter, the various types of nanoparticles, their properties, as well as their applications are presented briefly. Additionally, a short literature review with regard to the loading and release of drugs from nanoparticles is presented. The second chapter refers to the techniques and methods that were utilized in the context of the present thesis, as well as to the experimental procedures. Finally, in the third chapter the experimental results are presented and discussed. Based on the results of this study:  The magnetic nanocarriers based on copolymers poly(methacrylic acid)- graft -poly(ethyleneglycolmethacrylate) (p(MAA -g- EGMA)) have satisfying characteristics of size and z potential for long blood residence time after an intravenous injection, which is a prerequisite for their application as controlled (targeted) delivery systems for anticancer drugs. The high values of doxorubicin loading without stability loss is an important advantage.  The increase in the release rate of doxorubicin by the nanocarriers in albumin solutions with low pH (5-6) is important, since it facilitates a relatively selective release of the drug in cancer tumors which display lower pH than that of the normal tissues. In conclusion, the results of the research justify the further in-vitro study of the suitability of the magnetic doxorubicin nanocarriers as selective delivery systems of the drug to the cancer tumors.

Current diagnostic and therapeutic nanocarriers, including liposomes, micelles, and polymeric- and protein-based nanoparticles, are designed to have key functional properties such as: (i) longevity in the bloodstream, leading to accumulation of therapeutic cargos in neoplastic areas with leaky vasculatures; (ii) targeting of specific pathological sites through surface modification with targeting ligands; (iii) stimuli-responsive characteristics for controlled drug release under specific conditions. While some of these drug delivery systems have advanced into clinical stages, other nanocarriers remain under development to overcome issues with effective delivery such as lack of target-ability and fast clearance from circulation. Self-assembling peptides have recently shown great potential as nanocarrier materials for drug and gene delivery, owing to their safety, efficiency, and targeting capabilities. An amino acid pairing strategy enables us to design self/co-assembling peptides with multiple functionalities to fulfill drug delivery requirements. This thesis focuses on functionalization and characterization of self/co-assembling peptides as nanocarriers for hydrophobic anticancer drug delivery. Diethylene glycol (DEG) conjugation and protein binding are the two modification strategies used in this thesis to impart longevity and target-ability upon the peptide-based delivery system. The studies include: (i) characterization of self-assembling properties of the diethylene glycol (DEG)-conjugated amino acid pairing peptide AAP8, (ii) investigation of the self/co-assembling features of a model ionic-complementary peptide (EAR8-II) in complex with the hydrophobic drug pirarubicin, and the anticancer activity of the complex, (iii) the interactions between peptide-drug complexes and serum proteins from the thermodynamic viewpoint, (iv) quantification of the effect of protein binding to the peptide-based delivery system on immune responses and biocompatibility, and (v) exploration of the targeting capability of albumin-bound peptide-drug complexes towards lung cancer cells. Uncontrollable aggregation of AAP8 was the first issue to address in order to develop a promising platform for the peptide-based delivery system. Diethylene glycol (DEG), a short segment of polyethylene glycol (PEG), was conjugated to AAP8 either at one or both terminals, and then self-assembling and drug encapsulation properties of both functionalized AAP8s were characterized to evaluate the effect of DEG-modification. The results illustrated a significant reduction in uncontrollable aggregation, and the formation of uniform fibular nanostructures. In addition, DEG conjugation provided the peptide with safer features towards immune cells by reducing cellular toxicity to macrophages. Moreover, DEG-functionalization improved hydrophobic drug stabilization, as demonstrated by sustained cytotoxic efficacy against lung carcinoma cells over a relatively long time compared to the non-functionalized AAP8. Protein binding strategy was the second approach to utilize the peptide-based delivery system with more biocompatibility and target-ability features. EAR8-II was studied as a model ionic-complementary peptide with high capability of pirarubicin encapsulation and anticancer activities against different cancer cells. Albumin as a most abundant protein in serum was selected to assess its binding affinity to the delivery system, and evaluate its binding effect on immune responses and anticancer activities. The results showed a central role of albumin in the in vitro delivery of peptide-drug complexes to target lung cancer cells based on the following characteristics: (a) Non-covalent binding of albumin to the complex through hydrogen bonding and Van der Waals interactions. The interaction was confirmed by physicochemical methods such as fluorescence quenching and isothermal titration calorimeter (ITC). (b) Shielding properties of albumin for the complex against macrophages and blood components (erythrocytes and complement protein C5b-9). In the presence of albumin, phagocytosis and cytokine expression level of macrophages and hemolytic activity of the peptide-drug complex reduced significantly due to the smaller particle size of the albumin-bound complexes compared to unprotected ones. (c) Targeting the lung cancer cells, possibly because of the inhibition of the albumin-binding protein SPARC (secreted protein, acidic and rich in cysteine). SPARC is a glycoprotein over expressed in lung cancer cells with high affinity to albumin. The results from in vitro SPARC expression in A549 cells, a type of human non-small cell lung carcinoma (NSCLC), showed a significant drop by the albumin-bound complex at the mRNA level evaluated by qRT-PCR. This effect can be explained by transporting the albumin-bound complex into the cell surface, binding to the SPARC proteins, and so inhibiting the SPARC expressions. This work lays out a foundation for modification and characterization of the self/co-assembly peptide-based nanocarriers for hydrophobic anticancer drug delivery, especially to improve longevity and target-ability properties.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The aim of this study was to determine the bactericidal and bacteriostatic activity of a medicinal plant, Melaleuca alternifolia, in different formulation: oil free , essential oil associated with nanocarriers (nanoemulsion and nanocapsule), besides the associations between free oil and nanoformulations, as well as the antimicrobial activity of its major compound, terpinen - 4 - ol , in Rodococcus equi isolates (n = 24 ) from different sources. In order to determine the minimal inhibitory (MIC) and the minimum bactericidal (MBC) concentrations the microdilution method in Mueller-Hinton broth was employed in this study. The values of MIC and MBC ranged from 312.5 mg/mL to 2.500μg/mL. The results obtained with essential oil of M. alternifolia demonstrated low activity in oil free presentation; however, when it was associated with nanocarriers, its antimicrobial activity was enhanced, showing bacteriostatic activity. It was possible to verify that against R. equi isolates, both associations as well the major compound tested, showed both bactericidal and bacteriostatic activities. It is suggested that M. alternifolia, in association with nanocarriers systems, as well as terpinen -4-ol, presents potential for future studies concerning the equine rodococosis therapy. / O objetivo deste estudo foi determinar a atividade bactericida e bacteriostática da planta medicinal Melaleuca alternifolia em diferentes formulações: óleo livre; óleo essencial associado aos nanocarreadores (nanoemulsão e nanocápsula); além das associações entre o óleo livre e as nanoformulações; bem como, a atividade antimicrobiana do seu composto majoritário, o terpinen- 4-ol, em isolados de Rodococcus equi (n=24) oriundos de diferentes origens. Empregou-se o método de microdiluição em caldo Müeller-Hinton para a determinação das concentrações inibitória mínima (CIM) e bactericida mínima (CBM). Obteve-se CIM e CBM entre as concentrações 312,5μg/mL até 2.500μg/mL. Verificou-se que o óleo essencial de M. alternifolia possui baixa atividade quando em óleo livre; porém, quando associado aos nanocarreadores, sua atividade antimicrobiana foi potencializada, mostrando atividade bacteriostática. Evidenciou-se que frente aos isolados de R. equi, tanto as associações quanto o composto majoritário testados, demonstraram atividade bactericida e bacteriostática. Sugere-se que M. alternifolia em associação aos sistemas nanocarreadores, bem como o terpinen-4-ol, apresentam potencial para futuras pesquisas envolvendo o tratamento da rodococose equina.

L'artémisinine (ART) est un composé antipaludique majeur très actif sur les souches multi-résistantes de Plasmodium falciparum. Cependant, son application clinique est limitée par sa faible solubilité en milieu aqueux et sa faible biodisponibilité par voie orale. La mise en œuvre de cyclodextrines (CDs) bioestérifiées et de dérivés amphiphiles polyoxyéthylénés permet de viser deux objectifs : i) d'une part d'améliorer la concentration aqueuse d'ART à travers son association aux vecteurs colloïdaux de CDs modifiées et ii) d'autre part d'améliorer sa biodisponibilité et son efficacité thérapeutique à travers la décoration de la surface des vecteurs. La décoration surfacique est sensée augmenter le temps de circulation sanguine des nanovecteurs de manière à maintenir des doses plasmatiques efficaces d'ART sur une longue période, suite à une administration intraveineuse. Des suspensions colloïdales stables suffisamment chargées d'ART ont été mises au point permettant de palier le problème d'insolubilité en milieu aqueux de la molécule active et donc d'envisager son administration par voie parentérale. Les essais de lyodisponibilité indiquent que l'ART est libérée des nanosystèmes pendant une période de 4 jours pour les nanoréservoirs et de 11 jours pour les nanosphères. En plus des caractérisations physico-chimiques et pharmacotechniques, les potentialités des nanoparticules décorées en surface ont été évaluées et comparées au cours de tests biologiques. In vitro, l'ART mise en forme a montré une bonne efficacité sur des souches plasmodiales choloroquino-sensibles (3D7) et chloroquino-résistantes (K1) avec des CI50 très faibles de l'ordre de 3 à 6 ng/mL. Le concept de co-nano-assemblage de dérivés amphiphiles de γ-CD-C10 bioestérifiée et de polyéthylène glycol (PEG) dans les conditions de nanoprécipitation semble être une approche intéressante pour conférer des propriétés de furtivité aux nano-systèmes de γ-CD-C10. En effet, les études in vitro mettent en évidence une diminution significativement de la phagocytose par les cellules macrophagiques et/ou de l'adsorption des protéines du complément sérique à la surface des nanoparticules de γ-CD-C10 décorées par le polysorbate 80, le stéarate de PEG1500, et le DMPE-mPEG2000. In vivo, nous observons une augmentation du temps de circulation sanguine des nanoréservoirs γ-CD-C10/polysorbate 80 et des nanosphères γ-CD-C10/DMPE-mPEG2000. Enfin, les études de pharmacocinétique réalisées chez le rat montrent que les paramètres pharmacocinétiques de l'ART sont améliorés lorsqu'elle est associée aux deux systèmes nanoparticulaires précédents. En effet, des valeurs de clairance plasmatique très faibles et de temps de demi-vie plasmatique longs (3 et 5 heures, respectivement) de l'ART ont été enregistrées avec ces deux formulations. Ces formes vectorisées d'ART mises au point ouvrent de perspectives intéressantes pour la prise en charge thérapeutiques des crises de paludisme sévère. / Artemisinin (ART), an endoperoxide sesquiterpene lactone antimalarial drug isolated from a Chinese medicinal herb (Artemisia annua), has a fast action against chloroquine-sensitive and chloroquine-resistant strains of P. falciparum, making it very effective in the treatment of multidrug-resistant malaria. However, its poor aqueous solubility, short half-life, and high first-pass metabolism limit its use in therapeutics. The purpose of the present study was to investigate the potential of self-assembled bio-transesterified CD-based nanocarriers as ART delivery systems for an intravenous route. The objective of our study was twofold: (i) to improve the ART dosage through its association with CD esters-based nanocarriers, (ii) to ensure a sufficient blood circulation time of the nanocarriers through their surface decoration, which is a prerequisite to reach infected erythrocytes after systemic administration. Stable colloidal suspensions with good ART association rate were developed to solve the problem of insolubility in aqueous medium of the active molecule and therefore consider its parenteral administration. The γ-CD-C 10 based nanospheres showed a significant sustained release profile of ART extended up to 4 days for nanosphères and 11 days for nanoreservoirs. In addition to the physicochemical characterizations, the potential of nanoparticles decorated on the surface were evaluated and compared in biological tests. The results from this study indicate that ART-loaded nanosystems, mainly PEGylated ones exhibit satisfactory in vitro activity against Chloroquine-sensitive (3D7) and chloroquine-resistant (K1) strains of P. falciparum with very low IC50 of the order of 3 to 6 ng / mL. The concept of co-assembly of nano-amphiphilic derivatives of γ-CD-C10 bioestérifiée and polyethylene glycol (PEG) under conditions of nanoprecipitation seems to be an interesting approach to impart stealth nano-systems-γ-CD C10. Indeed, in vitro studies show a significant decrease in phagocytosis by macrophages and/or adsorption of serum complement proteins on the surface of nanoparticles of γ-CD-C10 decorated by polysorbate 80, PEG1500 stearate, and mPEG2000-DMPE. In vivo, we observed an increase in blood circulation time of nanoreservoirs γ-CD-C10/polysorbate 80 and nanospheres γ-CD-C10/DMPE-mPEG2000. Finally, pharmacokinetic studies in rats show that the pharmacokinetics of ART are enhanced when combined with previous two nanoparticle systems. Indeed, values of plasma clearance and very low time of long plasma half-life (3 to 5 hours, respectively) of ART were recorded with both formulations. These colloidal forms of ART developed open up interesting prospects for the therapeutic treatment of severe malaria.

A fosfoetanolamina sintética (FO-S), um fosfomonoéster, apresenta relevante atividade antitumoral. Contudo, a utilização de um carreador para encapsular a FO-S em lipossomas poderia favorecer a sua disponibilidade no microambiente tumoral, possibilitando o aumento da sua eficácia. Desta forma, o presente estudo avaliou a eficiência de encapsulamento da FO-S em lipossomas de DODAC e o seu potencial antitumoral. Os lipossomas foram preparados por ultrasonicação e caracterizados físicoquimicamente. A citotoxidade foi avaliada nas linhagens tumorais B16F10 (melanoma murino), Hepa1c1c7 (hepatocarcinoma murino) e Skmel-28 (melanoma humano) e nas células normais HUVEC, após o tratamento com diferentes concentrações dos lipossomas DODAC/FO-S, no tempo de 24 horas. A internalização dos lipossomas e o potencial elétrico mitocondrial foram analisados por microscopia confocal a laser. Adicionalmente, a expressão das proteínas caspases 3 e 8 ativas, receptor DR4, citocromo c, p53, p21, Bax, p27, CD44, CD90, Bcl-2 e ciclina D1 foi quantificada por citometria de fluxo. Para os estudos in vivo, os camundongos C57BL/6J portadores de hepatocarcinoma foram tratados com FO-S, DODAC/FO-S e DODAC, pelas vias intraperitoneal (IP) e intrahepática (IH), durante 20 dias. Os resultados demonstraram que os lipossomas apresentaram aspecto esférico e alta eficiência de encapsulação da FO-S, como também promoveram maior citotoxicidade nas linhagens tumorais estudadas, em comparação com FO-S. Além disto, nas células B16F10 e Hepa1c1c7, ocasionou parada nas fases S e G2/M do ciclo celular. A linhagem Hepa1c1c7 foi a mais sensível ao tratamento com os lipossomas DODAC/FO-S, os quais foram internalizados em até 6 horas e promoveram a diminuição de CD90, CD44, ciclina D1 e Bcl-2, o aumento de p53, p21, p27, Bax e caspases 8 e 3 ativas e a liberação do citocromo c. O aumento significativo das caspases 8 e 3 ativas, expressão do receptor DR4 e a liberação do citocromo c também ocorreu nas linhagens B16F10 e Skmel-28. Os resultados in vivo mostraram que os lipossomas DODAC/FO-S e a FO-S não induziram hepatotoxicidade, nefrotoxicidade e caquexia. Os lipossomas DODAC/FO-S não ocasionaram mielossupressão e hemólise, apresentando menor toxicidade em relação a FO-S, administrada pelas vias IP e IH. Além disto, os tratamentos com DODAC/FO-S (IH) e FO-S (IH e IP) foram efetivos em diminuir o número de células na fase S. Contudo, apenas os lipossomas DODAC/FO-S (IH) reduziram significamente os focos tumorais, aumentando as áreas de necrose, promovendo também o aumento da expressão gênica da p53, ciclina B1 e caspases 8 e 3. O conjunto dos resultados in vivo e in vitro demonstraram que a formulação lipossomal DODAC/FO-S foi capaz de maximizar os efeitos antitumorais da FO-S, ativando as vias intrínsecas e extrínsecas da apoptose / Synthetic phosphoethanolamine (PHO-S) - a phosphomonoester - has shown relevant anticancer effects. However, the utilization of a carrier to encapsulate the PHOS in liposomes can maximize its availability in the tumor microenvironment, allowing an increase in its effectiveness. Thus, the present study has evaluated efficiency of PHO-S encapsulation in DODAC liposomes and its antitumor potential. The liposomes were prepared by ultrasonication and physico-chemically characterized. The cytotoxic effects were evaluated on B16F10 cells (murine melanoma), Hepa1c1c7 cells (murine hepatocellular carcinoma), Skmel-28 (human melanoma) and in endothelial cells HUVEC, after treatment with DODAC/PHO-S liposomes at different concentrations for 24 hours. The internalization of the liposomes and mitochondrial electrical potential were analyzed by confocal laser microscopy. Additionally, the expression of active caspases 3 and 8, receptor DR4, cytochrome c, p53 p53, p21, Bax, p27, CD44, CD90, Bcl-2 and cyclin D1 proteins was quantified by flow cytometry. For in vivo studies, C57BL/6J mice with hepatocellular carcinoma were treated with PHO-S, DODAC/PHO-S and DODAC, by intraperitoneal (IP) and intratumoral (IT) routes for 20 days. The results demonstrated that liposomes presented spherical aspect and high PHO-S encapsulation efficiency, as also promoted high cytotoxic effect - compared with PHO-S. Furthermore, in B16F10 and Hepa1c1c7 cells, the liposomes induced S and G2/M cell cycle arrest. Hepa1c1c7 cells showed greater sensitivity to the DODAC/PHO-S formulation, which were internalized until 6 hours and promoted a decrease in the expression of CD90, CD44, cyclin D1 and Bcl-2, an increase of de p53, p21, p27, Bax and active caspases 8 and 3 and the liberation of cytochrome c. The significant increase in the expression of active caspases 3 and 8, DR4 receptor and liberation of cytochrome c also occurred in B16F10 and Skmel-28 cells. In vivo results showed that DODAC/PHO-S liposomes and PHO-S did not induce nephrotoxicity, hepatotoxicity and cachexia. DODAC/PHO-S liposomes did not cause myelosuppression and hemolysis, presenting lower toxicity in relation to PHO-S - when administered by IP and IT routes. Moreover, treatment with DODAC/PHO-S (IT) and PHO-S (IT and IP) effectively decreased the number of cells in S phase. However, only DODAC/PHO-S liposomes significantly reduced the number of tumor foci, increasing area of necrosis, and also promoting an increase in gene expression of p53, cyclin B1 and caspases 8 and 3. The set of in vitro and in vivo results demonstrated that DODAC/PHO-S liposomal formulation was capable of maximizing the PHO-S antitumor effects, activating the intrinsic and extrinsic pathways of the apoptosis

Orientador: Oswaldo Luiz Alves / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-21T13:14:24Z (GMT). No. of bitstreams: 1 Paula_AmauriJardimde_D.pdf: 5147163 bytes, checksum: 5adb7ae3c081749f414b5e64e35a8135 (MD5) Previous issue date: 2012 / Resumo: Partindo-se do método de Stöber, um elegante e eficiente processo de síntese de partículas coloidais de SiO2, adaptações foram feitas para que fosse possível produzir nanopartículas porosas (50-80 nm) com alto valor de área de superfície (~1000 m g), volume de poros (~1,5 m g) e alta estabilidade coloidal. As nanoestruturas são compostas de nanopartículas coloidais de sílica hierarquicamente funcionalizadas, com poros internos com estrutura desordenada e diâmetros que vão de 1,8 a 10 nanômetros, funcionalizados com grupos fenil, e superfície externa recoberta com grupos propilmetilfosfonato ionizáveis. A funcionalização hierárquica e quimicamente antagônica (hidrofóbica = poros internos; hidrofílica = superfície externa) permite que moléculas hidrofóbicas (baixa solubilidade em água) sejam facilmente incorporadas nas cavidades porosas hidrofóbicas, ao passo que as partículas se mantêm de forma estável dispersas em água por meses. Moléculas hidrofóbicas foram incorporadas pelas nanopartículas porosas de SiO2 em concentração de até 3% (m/m) através da mistura de suspensões coloidais aquosas desses nanomateriais e as moléculas insolúveis (pós). A capacidade de incorporação da molécula hidrofóbica em questão (camptotecina) foi significantemente maior que outros sistemas porosos de SiO2 que estão sendo atualmente usados. A eficiência dos veículos moleculares foi comprovada através do carreamento da camptotecina, um potente agente antitumoral que levou à inibição do crescimento de células leucêmicas humanas. Além disso, as abordagens sintéticas usadas nessa Tese também possibilitaram a funcionalização da superfície externa das nanopartículas com outros grupos orgânicos hidrofílicos e reativos, como o propilamina. Consequentemente, as características dessas nanopartículas de SiO2 aqui mostradas preenchem uma série de demandas científicas atuais: a necessidade de nanoestruturas porosas de sílica com ampla distribuição de tamanho de poro, com morfologia homogênea, estreita distribuição de tamanhos e com real dispersibilidade em água (coloidais). Assim, o conjunto de propriedades apresentado abre perspectivas envolvendo o uso desse sistema como uma plataforma tecnológica suscetível a várias aplicações, servindo como um veículo para dispersão e liberação de moléculas hidrofóbicas em meio aquoso / Abstract: Based on the Stöber method, an elegant and efficient process for synthesizing SiO2 colloidal nanoparticles, modifications were done in order to produce porous nanoparticles (50-80 nm) with high surface area (~1000 mg), volume of pores (~1,5 m g) and high colloidal stability. The nanostructures are made of hierarchically functionalized colloidal silica nanoparticles, with internal pores with disordered structure and diameters ranging from 1.8 to 10 nanometers, functionalized with phenyl groups; and external surface covered with ionizable propylmethylphosphonate groups. The hierarchical and chemically antagonistic functionalization (hydrophobic = internal pores; hydrophilic = external surface) allows hydrophobic molecules (low solubility in water) to be easily incorporated in the hydrophobic porous cavities, whereas particles maintain stably dispersed in water for months. Hydrophobic molecules were incorporated by the porous SiO2 nanoparticles in concentrations up to 3% (w/w) simply by mixing colloidal aqueous suspensions of these nanomaterials and insoluble molecules (powders). The uptake capacity for a specific hydrophobic molecule (camptothecin) was significantly higher than in other porous systems of SiO2 that have been currently used. The efficiency of the molecular vehicles was evidenced through the transportation of camptothecin, a potent antitumoral agent which led to the growth inhibition of human leukemic cells. Besides, the synthetic approach used in this thesis also made possible the functionalization of the external surface of nanoparticles with other hydrophilic and reactive organic groups, such as propylamine. Consequently, the characteristics of these SiO2 nanoparticles here shown fulfill several current scientific demands: necessity of porous silica nanostructures with a wide distribution of pore sizes, homogeneous morphology, narrow size distribution and real dispersibility in water (colloidal). Thereby, this set of properties opens up perspectives involving the use of this system as a technological platform susceptible to several applications, acting as a vehicle for the dispersion and liberation of hydrophobic molecules in aqueous media / Doutorado / Quimica Inorganica / Doutor em Ciências

Currently available anticancer therapies are inadequate and spur demand for improved technologies. Among others, the utilization of nanocarriers for anticancer drug delivery has shown great potential in cancer treatment. Nanocarriers can improve the therapeutic efficiency of the drugs with minimization of the undesirable side effects. To evaluate potential application of this technology, two forms of nanocarriers have been studied: multi-walled carbon nanotubes (MWCNTs) and apoferritin. The aim of this study was to determine, whether given cytostatics (ellipticine, etoposide and doxorubicin) are bound to these nanotransporters and how are they released from them, especially depending on pH. Since the pH of the tumor cells is lower than the pH of healthy cells it would be preferred that the drugs would release from nanocarriers at the lower pH while at the physiological pH the release of the drug would be eliminated. The results found show that ellipticine is actually released from its MWCNT- and apoferrtin-encapsulated form at acidic pH (5.0), while at pH 7.4 its interaction with nanocarriers is stable. Ellipticine released from MWCNT is activated by microsomal enzymes to reactive metabolites (13- hydroxyellipticine and 12-hydroxyellipticine) forming DNA adducts. The results indicate that both...